Well-defined oligomers of ubiquitin and ubiquitin-like polypeptides, and methods for preparing same

ABSTRACT

The present technology relates to well-defined oligomers comprising two or more monomers wherein each monomer is independently selected from a ubiquitin polypeptide or a ubiquitin-like polypeptide, and the monomers are covalently linked to each other via a thioether group or groups. Further provided are monomer building blocks and methods of making the monomers and oligomers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/624,201 filed Apr. 13, 2012, the entire disclosure of which ishereby incorporated by reference in its entirety.

BACKGROUND

Covalent attachment of ubiquitin (Ub) and ubiquitin-like proteins (Ubls)to the ε-amino group of lysine residues in a target protein (includingubiquitin itself), a process termed ubiquitination, is one of the mostprevalent mechanisms for regulating protein function and stability ineukaryotes. Indeed, sequence annotations suggest nearly 5% of the humangenome is dedicated to the coupling and removal of Ub/Ubls to and fromproteins. Given the central role of the ub network in cellularphysiology, misregulation is often associated with numerous humandiseases including. e.g., cancer, immune disorders neurodegenerativediseases and congestive heart failure.

Ubiquitination is unique among the ensemble of posttranslationalmodifications (PTMs), specifically from the standpoint of signaldiversity. For example, in contrast to other prevalent PTMs such asphosphorylation, proteins can be modified with Ub on a single lysineresidue (monoUb), multiple lysines (multi-monoUb), or a single lysinewith a polymeric chain of Ub (polyUb). With regards to polyUb chainformation, Ub possesses seven lysine residues (K6, K11, K27, K29, K33,K48, and K63), each of which may form an isopeptide linkage with thecarboxy terminus of another Ub molecule. This feature adds significantcomplexity to intracellular Ub signaling networks as it permits theassembly of chains with many different types of linkages and lengthswith the potential to control distinct biological processes.

A number of reports have recently emerged describing chemical approachesto the site-specific conjugation of Ub molecules through nativeNε-Gly-L-Lys isopeptide linkages as well as various normative linkages.Indeed, some of the methods have elucidated important structuraldistinctions for Ub dimers linked through the different Ub lysines, andenabled studies that uncovered how the structure and function of targetproteins is altered upon Ub modification. However, many of the chemicalapproaches designed to recapitulate the Nε-Gly-L-Lys linkage suffer fromdrawbacks such as instability, lengthy synthetic manipulations, and/orthe use of specialized recombinant DNA technologies for incorporatingunnatural amino acids. Moreover, branched Ub oligomers in which two ormore Ub molecules are covalently attached to a single Ub throughdifferent lysines appear to be inaccessible using known methods.

SUMMARY

The present technology provides well-defined oligomers of Ub and Ublpolypeptides constructed using thioether groups rather than thenaturally occurring isopeptide linkages. The thioether groups can bedesigned to closely mimic the native isopeptide or may be varied. Alsoprovided are the Ub and Ubl building blocks, i.e., monomers, forconstructing the oligomers, including Ub and Ubl polypeptides withcarboxy terminal alkenes. Methods of preparing the monomers are alsodisclosed. The present technology further includes methods of couplingthe monomers to efficiently and precisely form the Ub and Ubl oligomers.Oligomers of the present technology may be used to probe the roles ofthe analogous natural Ub and Ubl oligomers in cellular physiology andhuman disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. High resolution FT-ICR MS analysis of intact full-length Ub-AA(M¹⁰⁺ charge state is shown), according to the Examples. Circlesrepresent the theoretical isotopic distribution. Calc'd and Expt'l referto the calculated and experimental molecular weights of full lengthUb-AA, respectively.

FIG. 2. Construction of K48C-linked Ub₂ using TEC, according to theExamples: (A) Reaction scheme for the TEC of Ub-AA and UbK48C (PDB codefor Ub structure shown: 1UBQ). (B) Structures of the free-radicalinitiators used in this study. (C) Coomassie-stained SDS-PAGE analysisof TEC reactions carried out with different initiators. Each lanerepresents a reaction conducted with Ub-AA (1 mM) and UbK48C (1 mM), andfree-radical initiator LAP (0.1 mM) or V-50 (100 mM) at pH 5.0. In thecase of LAP, the reactions were irradiated with long wavelength light at365 nm. Black dot indicates presence of specified reaction component.(D) SDS-PAGE analysis of TEC reactions with varying concentrations ofthe LAP photoinitiator.

FIG. 3. Coomassie-stained SDS-PAGE analysis of TEC reactions with allseven UbKxC nmtants, according to the Examples. Dimers are observed inall reactions containing Ub-AA. The higher MW bands observed in thereactions conducted with Ub-AA are present in differing amountsdepending on the UbKxC mutant.

FIG. 4. Representative purification of TEC products: FPLC chromatogramfor the K48C-linked Ub dimer, according to the Examples. The inset showsCoomassie-stained SDS-PAGE analysis for each peak observed in thechromatogram: peak 4 contains the purified K48C-linked Ub dimer. MSanalysis of peak 3 corresponds to the mass of Ub-AA plus the phosphinateportion of the LAP photoinitiator (for more details, see below).

FIG. 5. High resolution FT-ICR MS analysis of crude TEC reactions usingintact full-length proteins, according to the Examples. Wide view showsabundance of Ub dimers in comparison to the starting materials UbKxC andUb-AA (M¹⁰⁺ charge state for starting materials, M²⁰⁺ charge state fordimer is shown). Zoom in shows each dimer compared to the theoreticalisotopic distribution (dots above peaks).

FIG. 6. High resolution FT-ICR MS analysis of each purified dimer,according to the Examples. Circles represent theoretical isotopicabundance distribution of the isotopomer peaks. Calc'd: calculated mostabundant molecular weight. Expt'l: experimental most abundant molecularweight.

FIG. 7. FT-ICR analysis of each purified branched trimer, according tothe Examples. Circles represent theoretical isotopic abundancedistribution of the isotopomer peaks. Calc'd: calculated most abundantmolecular weight. Expt'l: experimental most abundant molecular weight.

FIG. 8. ECD analysis of K63C-linked dimer, according to the Examples:(A) K63C-linked Ub₁₋₇₄ GlyGly-AA parent ion isolation (M⁹⁺ charge state)with insert of isotopomers. (B) Map of observed fragments. Data analysisfor the map on top includes Nε-Gly-L-homothiaLys thioether linkermodification at cysteine-63 (red) in c and z* ion predictions. Bottommap does not include thioether linker modification in theoreticalanalysis. (C) Key ECD fragment ions for mapping thioether linkage siteon UbK63C. Circles represent theoretical isotopic abundance distributionof the isotopomer peaks. Calc'd: calculated most abundant molecularweight. Expt'l: experimental most abundant molecular weight.

FIG. 9. ECD analysis of K6C-linked dimer, according to the Examples: (A)K6C-linked Ub₁₋₇₄ GlyGly-AA parent ion isolation (M¹⁰⁺ charge state)with insert of isotopomers. (B) Map of observed fragments. Data analysisfor the map on top includes Nε-Gly-L-homothiaLys thioether linkermodification at cysteine-6 (red) in c and z* ion predictions. Bottom mapdoes not include thioether linker modification in theoretical analysis.(C) Key ECD fragment ions for mapping thioether linkage site on UbK6C.Circles represent theoretical isotopic abundance distribution of theisotopomer peaks. Calc'd: calculated most abundant mass. Expt'l:experimental most abundant mass.

FIG. 10. ECD analysis of K48C-linked Ub dimer, according to theExamples: (A) K48C-linked Ub₁₋₇₄ GlyGly-AA parent ion isolation (M⁹⁺charge state) with insert of isotopomers. (B) Map of observed fragments.Data analysis for the map on top includes Nε-Gly-L-homothiaLys thioetherlinker modification at cysteine-48 (red) in c and z* ion predictions.Bottom map does not include thioether linker modification in theoreticalanalysis.

FIG. 11. ECD analysis of K6C, K48C-linked branched Ub trimer, accordingto the Examples: (A) K6C, K48C Ub₁₋₇₄ GlyGlyAA₂ parent ion isolation(M¹⁰⁺ charge state) with insert of isotopomers. (B) Map of observedfragments. Data analysis includes Nε-Gly-L-homothiaLys thioether linkermodification at cysteine-6 and cysteine-48 (red) in c and z* ionpredictions.

FIG. 12. Key ECD fragment ions for K6C, K48C-linked trimer, according tothe Examples. Circles represent theoretical isotopic abundancedistribution of the isotopomer peaks. Calc'd: calculated most abundantmolecular weight. Expt'l: experimental most abundant molecular weight.

FIG. 13. ECD analysis of K11C, K48C-linked trimer, according to theExamples. (A) K11C, K48C-linked Ub₁₋₇₄ GlyGlyAA₂ parent ion isolation(M¹⁰⁺ charge state) with insert of isotopomers. (B) Map of observedfragments. Data analysis for the map on the top includes theNε-Gly-L-homothiaLys thioether linker modification at cysteine-11 andcysteine-48 (red) in c and z* ion predictions. Bottom map does notinclude thioether linker modifications in the sequence.

FIG. 14. Key ECD fragment ions for K11C, K48C-linked trimer. Circlesrepresent theoretical isotopic abundance distribution of the isotopomerpeaks. Calc'd: calculated most abundant molecular weight. Expt'l:experimental most abundant molecular weight.

FIG. 15. ECD analysis of K48C, K63C-linked trimer, according to theExamples. (A) K48C, K63C-linked Ub₁₋₇₄ GlyGlyAA₂ parent ion isolation(M¹⁰⁺ charge state) with insert of isotopomers. (B) Map of observedfragments. Data analysis for the map on top includes theNε-Gly-L-homothiaLys thioether linker modification at cysteine-48 andcysteine-63 (red) in c and z* ion predictions. Bottom map does notinclude thioether linker modifications in theoretical analysis.

FIG. 16. Key ECD fragment ions for K48C, K63C-linked trimer, accordingto the Examples. Circles represent theoretical isotopic abundancedistribution of the isotopomer peaks. Calc'd: calculated most abundantmolecular weight. Expt'l: experimental most abundant molecular weight.

FIG. 17. High resolution FT-ICR MS analysis of K27C-linked dimer withvarying amounts of Ub-AA, according to the Examples. Relative amounts ofUb-AA to UbK27C are shown on the right. Box shows where M²⁰⁺ dimershould appear.

FIG. 18. FT-ICR analysis of K29C-linked dimer with varying amounts ofUb-AA, according to the Examples. The purple box highlights theformation of the desired dimer. Relative concentrations of Ub-AA toUbK29C are shown on the right.

FIG. 19. High resolution FT-ICR MS analysis of K33C-linked dimer withvarying amounts of Ub-AA, according to the Examples. The purple boxhighlights the formation of the desired dimer. Relative concentrationsof Ub-AA to UbK33C are shown on the right.

FIG. 20. Hydrolytic cleavage of K11C, K48C-linked Ub trimer with IsoT,A20-OUT, and AMSH, according to the Examples.

FIG. 21. Hydrolytic cleavage of K48C, K63C-linked Ub trimer with IsoT,A20-OUT, and AMSH, according to the Examples.

FIG. 22. Hydrolytic cleavage of K6C, K48C-linked Ub trimer with IsoT,A20-OUT, and AMSH, according to the Examples.

FIG. 23. Hydrolytic cleavage of K6C, K48C-linked Ub trimer with USP7,according to the Examples.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

The present technology is described herein using several definitions, asset forth throughout the specification. As used herein, unless otherwisestated, the singular forms “a,” “an,” and “the” include the pluralreference. Thus, for example, a reference to “a protein” is a referenceto one or more proteins.

Alkyl groups include straight chain and branched alkyl groups havingfrom 1 to 20 carbon atoms or, in some embodiments, from 1 to 12, 1 to 8,1 to 6, or 1 to 4 carbon atoms. Alkyl groups further include cycloalkylgroups. Examples of straight chain alkyl groups include those with from1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl,n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groupsinclude, but are not limited to, isopropyl, iso-butyl, sec-butyl,tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.Representative substituted alkyl groups may be substituted one or moretimes with substituents such as those listed below. For example, theterm haloalkyl refers to an alkyl group substituted with one or morehalogen atoms.

Alkenyl groups include straight and branched chain alkyl and cycloalkylgroups in which at least one double bond exists between two carbonatoms. Thus, alkenyl groups have from 2 to about 20 carbon atoms, andtypically from 2 to 12 carbons or, in some embodiments, from 2 to 8, 2to 6, or 2 to 4 carbon atoms. In some embodiments, alkenyl groupsinclude cycloalkenyl groups having from 4 to 20 carbon atoms, 5 to 20carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbon atoms.Examples include, but are not limited to vinyl, allyl, —CH═CH(CH₃),—CH═C(CH₃)₂, —C(CH₃)═CH₂, —C(CH₃)═CH(CH₃), —C(CH₂CH₃)═CH₂, cyclohexenyl,cyclopentenyl, among others. Representative substituted alkenyl groupsmay be mono-substituted or substituted more than once, such as, but notlimited to, mono-, di- or tri-substituted with substituents such asthose listed below.

The term “alkylene” alone or as part of another substituent refers to adivalent radical of an alkyl (including cycloalkyl) group. Each alkylenemay be divalent at the same carbon or different carbons. Thus, e.g., thealkylene group based on ethyl is ethylene, and includes —CH(CH₃)— aswell as —CH₂CH₂—. For alkylene groups, no particular pattern ofattachment or orientation of the group is implied.

Alkylene oxide is an alkylene group in which one or more carbon atomshave been replaced with oxygen such that the resulting group ischemically stable. Nonlimiting examples of alkylene oxide groups includepolyethylene glycol, polypropylene glycol, polytetramethylene oxide andthe like.

Aryl groups are cyclic aromatic hydrocarbons having 6-14 carbons andthat do not contain heteroatoms. Aryl groups include monocyclic,bicyclic and tricyclic ring systems. Thus, aryl groups include, but arenot limited to, phenyl, azulenyl, heptalenyl, biphenylenyl, indacenyl,fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl,chrysenyl, biphenyl, anthracenyl, indenyl, indanyl, pentalenyl, andnaphthyl groups. In some embodiments, aryl groups contain 6-12 carbons,and in others from 6-10 or even 6-8 carbon atoms in the ring portions ofthe groups. The phrase “aryl groups” further includes groups containingfused rings, such as fused aromatic-aliphatic ring systems (e.g.,indanyl, tetrahydronaphthyl, and the like). However, it does not includearyl groups that have other groups, such as alkyl or halo groups, bondedto one of the ring members. Rather, groups such as tolyl are referred toas substituted aryl groups. Representative substituted aryl groups maybe mono-substituted or substituted more than once. For example,monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-,5-, or 6-substituted phenyl or naphthyl groups, which may be substitutedwith substituents such as those listed below.

The term “arylene” or “aralkylene” alone or as part of anothersubstituent refers to a divalent radical of an aryl or aralkyl group.Each arylene or aralkylene will be divalent at different carbons of thearomatic ring or, in the case of aralkylene, may be divalent at the samecarbon or different carbons of the alkylene portion. Further thealkylene portion of aralkylene may be a single chain or two separatechains attached to different carbons of the aromatic group. Thus,examples of arylene and aralkylene include but are not limited tophenylene, benzylene, ethylphenylethylene and the like. No particularpattern of attachment or orientation of the arylene or aralkylene groupis implied.

In general, “substituted” refers to a group, as defined above (e.g., analkyl or aryl group) in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms. Substituted groups also include groups in which one or more bondsto a carbon(s) or hydrogen(s) atom are replaced by one or more bonds,including double or triple bonds, to a heteroatom. Thus, a substitutedgroup will be substituted with one or more substituents, unlessotherwise specified. In some embodiments, a substituted group issubstituted with 1, 2, 3, 4, 5, or 6 substituents. Examples ofsubstituent groups include: halogens (i.e., F, Cl, Br, and I);hydroxyls: alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy,heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls(oxo);carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines;aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls;sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones;azides; amides; ureas; amidines; guanidines; enamines; imides;isocyanates; isothiocyanates; cyanates; thiocyanates; inines; nitrogroups; nitriles (i.e., CN); and the like.

As used herein, the terms “sequence identity” or percent “identity”,when used in the context of two or more nucleic acids or polypeptidesequences, refers to two or more sequences or subsequences that are thesame or have a specified percentage of amino acid residues ornucleotides that are the same (i.e., about 60% identity, preferably 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, orhigher identity over a specified region (e.g., nucleotide sequenceencoding an antibody described herein or amino acid sequence of anantibody described herein), when compared and aligned for maximumcorrespondence over a comparison window or designated region) asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection (See, e.g., NCBI web site). Such sequences are then said tobe “substantially identical.” This term also refers to, or can beapplied to, the complement of a test sequence. The term also includessequences that have deletions and/or additions, as well as those thathave substitutions. Typically, identity exists over a region that is atleast about 25 amino acids or nucleotides in length, or more preferablyover a region that is at least 50-100 amino acids or nucleotides inlength.

As used herein, the terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to mean a polymer comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. Polypeptide refers to both short chains, commonly referred to aspeptides or oligomers, and to longer chains, generally referred to asproteins. Unless otherwise specified, the terms “polypeptide,”“protein,” and “peptide” also encompass various modified forms thereof.Such modified forms may be naturally occurring modified forms orchemically modified forms. Examples of modified forms include, but arenot limited to, glycosylated forms, phosphorylated forms, myristoylatedforms, palmitoylated forms, ribosylated forms, acetylated forms,ubiquitinated forms, etc. Modifications also include intra-molecularcrosslinking and covalent attachment to various moieties such as lipids,flavin, biotin, polyethylene glycol or derivatives thereof, etc. Inaddition, modifications may also include cyclization, branching andcross-linking. Further, amino acids other than the conventional twentyamino acids encoded by genes may also be included in a polypeptide.

As used herein, the terms “variant” or “mutant” are used to refer to aprotein or peptide which differs from a naturally occurring protein orpeptide (i.e., the “prototype” or “wild-type” protein) by modificationsto the naturally occurring protein or peptide, but which maintains thebasic protein and side chain structure of the naturally occurring form.Such changes include, but are not limited to: changes in one, few, oreven several amino acid side chains; changes in one, few or severalamino acids, including deletions (e.g., a truncated version of theprotein or peptide), insertions and/or substitutions; changes instereochemistry of one or a few atoms; and/or minor derivatizations,including but not limited to: methylation, glycosylation,phosphorylation, acetylation, myristoylation, prenylation, palmitation,amidation and/or addition of glycosylphosphatidyl inositol. A “variant”or “mutant” can have either enhanced, decreased, changed, orsubstantially similar properties as compared to the naturally occurringprotein or peptide. In one embodiment, a variant of Ub or Ubl is asubstrate for a deubiquitinating enzyme or for a ubiquitin bindingprotein.

As used herein, the term “oligomer” refers to a short polymer composedof two or more monomers, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, or 50 monomers or a range of monomers between and includingany two of these values.

As used herein, reference to an ubiquitin (Ub) protein or polypeptide orubiquitin-like (Ubl) protein or polypeptide, including an isolated Ub orUbl, includes full-length proteins, fusion proteins, or any fragment,mutant, variant, or homologue of such a protein. Such a Ub or Ublprotein can include, but is not limited to, purified Ub or Ubl protein,recombinantly produced Ub or Ubl protein, soluble Ub or Ubl protein,insoluble Ub or Ubl protein, and isolated Ub or Ubl protein associatedwith other proteins, and isolated Ub or Ubl associated with cellularmembranes. An isolated protein is a protein (including a polypeptide orpeptide) that has been removed from its natural milieu (i.e., that hasbeen subject to human manipulation) and can include purified proteins,partially purified proteins, recombinantly produced proteins, andsynthetically produced proteins, for example. As such, “isolated” doesnot reflect the extent to which the protein has been purified.Typically, an isolated Ub or Ubl polypeptide is produced recombinantly.

In addition, and by way of example, a “human Ub protein” refers to a Ubprotein from a human (Homo sapiens) or to a Ub protein that has beenotherwise produced from the knowledge of the structure (e.g., sequence)and perhaps the function of a naturally occurring Ub protein from Homosapiens. In other words, a human Ub protein includes any Ub protein thathas substantially similar structure and function of a naturallyoccurring Ub protein from Homo sapiens or that is a biologically active(i.e., has biological activity) homologue of a naturally occurring Ubprotein from Homo sapiens as described in detail herein. As such, ahuman Ub protein can include purified, partially purified, recombinant,mutated/modified and synthetic proteins. According to the presentinvention, the terms “modification” and “mutation” can be usedinterchangeably, particularly with regard to the modifications/mutationsto the amino acid sequence of Ub (or nucleic acid sequences) describedherein. The amino acid sequence of human Ub is 76 amino acids in lengthand is a post-translational modification of the full-lengthtranslational product of human Ub (Genbank Accession No. CAA44911.1).Human Ub is set forth below as SEQ ID NO: 1.

(SEQ ID NO: 1) 1 MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN61 IQKESTLHLV LRLRGG

A number of proteins also modify the E-amino group in proteinsanalogously to ubiquitin, but function in distinct signaling pathways.These proteins are known as ubiquitin-like proteins (Ubl). Oligomers ofUbl may be constructed as disclosed herein. Ubl include but are notlimited to SUMO1, SUMO2, SUMO3 and ISG15. Sequences of these proteinsare set forth below as SEQ ID NOS:2, 3, 4, and 5, respectively.

SUMO 1, GenBank Accession No. AAC50996.1 (SEQ ID NO: 2)  1 MSDQEAKPST EDLGDKKEGE YIKLKVIGQD SSEIHFKVKM TTHLKKLKES YCQRQGVPMN 61 SLRFLFEGQR IADNHTPKEL GMEEEDVIEV YQEQTGGHST VSUMO 2, GenBank Accession No. P61956.3 (SEQ ID NO: 3)  1 MADEKPKEGV KTENNDHINL KVAGQDGSVV QFKIKRHTPL SKLMKAYCER QGLSMRQIRF 61 RFDGQPINET DTPAQLEMED EDTIDVFQQQ TGGVYSUMO 3, GenBank Accession No. P55854.2 (SEQ ID NO: 4)  1 MSEEKPKEGV KTENDHINLK VAGQDGSVVQ FKIKRHTPLS KLMKAYCERQ GLSMRQIRFR 61 FDGQPINETD TPAQLEMEDE DTIDVFQQQT GGVPESSLAG HSFISG15, GenBank Accession No. AAH09507.1 (SEQ ID NO: 5)  1 MGWDLTVKML AGNEFQVSLS SSMSVSELKA QITQKIGVHA FQQRLAVHPS GVALQDRVPL 61 ASQGLGPGST VLLVVDKCDE PLNILVRNNK GRSSTYEVRL TQTVAHLKQQ VSGLEGVQDD121 LFWLTFEGKP LEDQLPLGEY GLKPLSTVFM NLRLRGGGTE PGGRS

Homologues can be the result of natural allelic variation or naturalmutation. A naturally occurring allelic variant of a nucleic acidencoding a protein is a gene that occurs at essentially the same locus(or loci) in the genome as the gene which encodes such protein, butwhich, due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Allelicvariants typically encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared. Oneclass of allelic variants can encode the same protein but have differentnucleic acid sequences due to the degeneracy of the genetic code.Allelic variants are well known to those skilled in the art.

Homologues can be produced using techniques known in the art for theproduction of proteins including, but not limited to, directmodifications to the isolated, naturally occurring protein, directprotein synthesis, or modifications to the nucleic acid sequenceencoding the protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis.

Homologues or variants of Ub or Ubl can be produced that contain one ormore conservative or non-conservative amino acid changes, compared withthe native enzyme, so long as the sterol binding or cholesterolincorporation activity is retained. Typically, variants have at least50%, at least 60%, at least 70%, at least 80%, or at least 90% aminoacid sequence identity compared to the original sequences such as anyone of SEQ ID NOs: 1, 2, 3, 4, or 5. In some embodiments, high sequenceidentity variants are provided in which the amino acid sequence identityof the variant to the Ub or Ubl is at least 95%, at least 96%, at least97%, at least 98% or even at least 99%. In other embodiments, Ub or Ublvariants include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more conservative ornonconservative amino acid substitutions such as 15, 20, 25, 30, or even40 amino acid substitutions so long as cholesterol incorporationactivity is retained. The ability of variants of Ub or Ubl to serve assubstrates for deubiquitination can be determined using a standardactivity assay, such as the assay described in the Examples.

Conservative variants can be obtained that contain one or more aminoacid substitutions of, e.g., SEQ ID NO: 1, in which an alkyl amino acidis substituted for an alkyl amino acid in the Ub or Ubl amino acidsequence, an aromatic amino acid is substituted for an aromatic aminoacid in Ub or Ubl amino acid sequence, a sulfur-containing amino acid issubstituted for a sulfur-containing amino acid in the Ub or Ubl aminoacid sequence, a hydroxy-containing amino acid is substituted for ahydroxy-containing amino acid in the Ub or Ubl amino acid sequence, anacidic amino acid is substituted for an acidic amino acid in the Ub orUbl amino acid sequence, a basic amino acid is substituted for a basicamino acid in the Ub or Ubl amino acid sequence, or a dibasicmonocarboxylic amino acid is substituted for a dibasic monocarboxylicamino acid in the Ub or Ubl amino acid sequence.

Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, (2) valine, leucine,and isoleucine, (3) phenylalanine, tyrosine, and tryptophan, (4)cysteine and methionine, (5) serine and threonine. (6) aspartate andglutamate, (7) glutamine and asparagine, and (8) lysine, arginine andhistidine.

Conservative amino acid changes in, e.g., the human Ub, can beintroduced by substituting appropriate nucleotides for the nucleotidesencoding SEQ ID NO: 1. Such “conservative amino acid” variants can beobtained, for example, by oligonucleotide-directed mutagenesis,linker-scanning mutagenesis, mutagenesis using the polymerase chainreaction, and the like. Ausubel et al., supra; Ausubel et al. (eds.),SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 5th Edition, John Wiley & Sons,Inc. (2002). Also see generally, McPherson (ed.), DIRECTED MUTAGENESIS:A PRACTICAL APPROACH, IRL Press (1991). A useful method foridentification of locations for sequence variation is called “alaninescanning mutagenesis” a described by Cunningham and Wells in Science,244:1081-1085 (1989).

Ub or Ubl variants that contain one or more non-conservative amino acidsubstitutions, such as substitution of cysteine residues for one or morelysine residues in Ub or Ubl having any one of SEQ ID NOs: 1, 2, 3, 4and 5 and that retain the ability to serve as a substrate fordeubiquitination enzymes can also be produced and used as disclosedherein. Other non-conservative amino acid substitutions are known in theart and include, without limitation, leucine for aspartate or valine forthreonine. Non-conservative variants can also include amino acidinsertions as compared to the native sequence such as, withoutlimitation, insertion of methionine. As will be appreciated by theskilled artisan, the same methods used for generating conservativevariants may be adapted and used to produce nonconservative variants.

In addition, routine deletion analyses of DNA molecules can be performedto obtain “functional fragments” of Ub, Ubl or homologues thereof. Thefragments are inserted into expression vectors in proper reading frame,and the expressed polypeptides are isolated and tested for the abilityto incorporate cholesterol into lipid bilayers. One alternative toexonuclease digestion is to use oligonucleotide-directed mutagenesis tointroduce deletions or stop codons to specify production of a desiredfragment. Alternatively, particular fragments of the Ub or Ubl gene canbe synthesized using the polymerase chain reaction. Standard techniquesfor functional analysis of proteins are described by, for example,Treuter et al., Molec. Gen. Genet., 240:113 (1993); Content et al.,“Expression and preliminary deletion analysis of the 42 kDa 2-5Asynthetase induced by human interferon,” in BIOLOGICAL INTERFERONSYSTEMS, PROCEEDINGS OF ISIR-TNO MEETING ON INTERFERON SYSTEMS, Cantell(ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGF Receptor,” inCONTROL OF ANIMAL CELL PROLIFERATION, Vol. 1, Boynton et al., (eds.)pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol. Chem.,270:29270 (1995); Fukunaga et al., J. Biol. Chem., 270:25291 (1995);Yanmaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meisel etal., Plant Molec. Biol., 30:1 (1996). In some embodiments the functionalfragment retains at least 50% or at least 60% of the amino acids of thenative sequence. In others the functional fragment retains at least 70%,at least 80%, at least 90%, at least 95%, at least 98% or at least 99%of the amino acids of the native sequence.

In one aspect, the present technology provides well-defined oligomers ofubiquitin and ubiquitin-like polypeptides. Such oligomers may be used astools for probing the manifold roles of ubiquitination in cellularphysiology and human diseases. The present oligomers include two or moremonomers wherein each monomer is independently selected from a ubiquitinpolypeptide or a ubiquitin-like polypeptide. The monomers are covalentlylinked to each other via a thioether group or groups. Typically, eachthioether group links the peptide backbone of one monomer to the carboxyterminus of another monomer. For example, each thioether group mayinclude a cysteine residue of one of the monomers. Thus, an oligomer maybe constructed wherein each monomer is a mutant in which each cysteineresidue in the thioether group replaces a lysine residue in the nativesequence of the monomer.

Oligomers of the present technology may include various numbers ofmonomers such as, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 monomers.The oligomers may be linear or branched. Linear oligomers, other thandimers, include one or more internal monomers that are each linked to 2monomers: one through a thioether group attached to the peptide backboneof the internal monomer and one through a thioether group attached tothe carboxy terminus of the internal monomer.

In contrast to linear oligomers, a branched oligomer includes at least 3monomers. At least one monomer (the internal monomer) of the branchedoligomer is covalently linked to at least 2 other monomers via thioethergroups to the peptide backbone of the internal monomer. In someembodiments, the thioether groups of at least two monomers includecysteines at different positions within the monomer. For example, forubiquitin, the internal monomer may include a thioether group at K6C anda thioether group at K48C, or at K11C and K48C, or at K48C and K63C.

Oligomers of the present technology may incorporate any Ub or Ublmonomers, including mutants, variants, homologues and fragments of anyof the foregoing. As described further below, the monomers may or maynot include an alkene attached to the carboxy terminus. In someembodiments, each monomer of the oligomer is independently selected froma polypeptide that

-   -   a. has at least 95% sequence identity to a sequence selected        from the group consisting of SEQ ID NOS:1, 2, 3, 4, and 5;    -   b. comprises a sequence that has at least 95% sequence identity        to a sequence selected from the group consisting of SEQ ID NOS:        1, 2, 3, 4, and 5; or    -   c. is a fragment of a polypeptide having at least 95% sequence        identity to a sequence selected from the group consisting of SEQ        ID NOS:1, 2, 3, 4, and 5, wherein the ubiquitin or        ubiquitin-like polypeptide is a substrate for a deubiquinating        enzyme or for proteins containing ubiquitin-binding domains,        (e.g., RPN10, RPN13, hHR23A, Dsk2, p62, NBR1, RAP80, NEMO, TAB2,        TAB3, and A20) when covalently attached via its carboxy terminus        to another protein.

Examples of mutant Ub and Ubl in which one or more lysine residues arereplaced with cysteine residues are set forth for SEQ ID NO:1 in Table 1and for SEQ ID NO:3 in Table 2. The asterisk (*) indicates presence ofthe specified mutation. Those skilled in the art will understand thatany of these mutants may be further modified to include a C-terminalalkene as described herein.

TABLE 1 Mutants of SEQ ID NO: 1 Mutant No. K6C K11C K27C K29C K33C K48CK63C 1 * 2 * 3 * 4 * 5 * 6 * 7 * 8 * * 9 * * 10 * * 11 * * 12 * * 13 * *14 * * 15 * * 16 * * 17 * * 18 * * 19 * * 20 * * 21 * * 22 * * 23 * *24 * * 25 * * 26 * * 27 * * 28 * * 29 * * * 30 * * * 31 * * * 32 * * *33 * * * 34 * * * 35 * * * 36 * * * 37 * * * 38 * * * 39 * * * 40 * * *41 * * * 42 * * * 43 * * * 44 * * * 45 * * * 46 * * * 47 * * * 48 * * *49 * * * 50 * * * 51 * * * 52 * * 53 * * * 54 * * * 55 * * * 56 * * *57 * * * 58 * * * 59 * * * 60 * * * 61 * * * 62 * * * 63 * * *

TABLE 2 Mutants of SEQ ID NO: 3 Mutant No. K11C K20C K32C K41C K44C 1 *2 * 3 * 4 * 5 * 6 * * 7 * * 8 * * 9 * * 10 * * 11 * * 12 * * 13 * *14 * * 15 * *

In another aspect, the present technology provides building blocks forconstructing the oligomers described herein. One such building block isa ubiquitin or ubiquitin-like polypeptide that includes a carboxyterminal alkenyl group. In some embodiments, a cysteine residue replacesat least one lysine residue in the ubiquitin or ubiquitin-likepolypeptide to provide a mutant Ub or Ubl bearing a C-terminal alkenegroup. As in the oligomers, the ubiquitin or ubiquitin-like polypeptidebuilding block may be a polypeptide that

-   -   a. has at least 95% sequence identity to a sequence selected        from the group consisting of SEQ ID NOS:1, 2, 3, 4, and 5;    -   b. comprises a sequence that has at least 95% sequence identity        to a sequence selected from the group consisting of SEQ ID        NOS:1, 2, 3, 4, and 5; or    -   c. is a fragment of a polypeptide having at least 95% sequence        identity to a sequence selected from the group consisting of SEQ        ID NOS:1, 2, 3, 4, and 5, wherein the ubiquitin or        ubiquitin-like polypeptide is a substrate for a deubiquinating        enzyme when covalently attached via its carboxy terminus to        another protein.

In some embodiments, the Ub or Ubl polypeptide building block is amutant in which each of 1, 2 or 3 lysine residues of the native sequenceis replaced with a cysteine residue. For example, the Ub or Ublpolypeptide building block may include 1, 2, or 3 mutations selectedfrom K6C, K11C, K27C, K29C, K33C, K48C, and K63C of Ub and K11C, K20C,K32C, K41C, and K44C of SUMO2. Specific mutants suitable for use as abuilding block for the present oligomers are set forth in Table 1 andTable 2 above.

The alkenyl group at the carboxy terminus of the Ub and Ubl polypeptidemay have a variety of structures to facilitate its attachment and/orcoupling to form oligomers For example, the carboxy terminal alkenylgroup of the Ub and Ubl polypeptide may have the structure:—NR¹(R²)CH═CH₂, wherein R¹ is H or C1-4 alkyl group; and R² is asubstituted or unsubstituted alkylene, alkylene oxide, arylene oraralkylene group. In some embodiments, R¹ is H. In others, R¹ is amethyl or ethyl group. In some embodiments, R² is unsubstituted, e.g,unsubstituted alkylene or alkylene oxide. In certain embodiments, thecarboxy terminal alkenyl group has the structure: —NH(CH₂)_(n)CH═CH₂,wherein n is an integer from 0 to 10. In certain embodiments, n is 1 or2 (i.e., allyl amine or butenyl amine). Aminoalkenes such as these maybe attached to the Ub and Ubl polypeptides by using Ub C-terminalhydrolases under conditions (a large stoichiometric excess ofaminoalkene) that promote amide bond formation rather than amide bondhydrolysis.

In still another aspect, the present technology provides a method ofmaking an oligomer as described herein using one or more Ub and/or Ublbuilding blocks as described herein. The method includes coupling afirst monomer to a second monomer under free radical conditions suchthat the first and second monomers are linked by a thioether group. Thefirst monomer is selected from a ubiquitin or ubiquitin-like polypeptidethat includes a carboxy terminal alkenyl group and the second monomer isselected from a ubiquitin or ubiquitin-like polypeptide comprising oneor more cysteine residues. Thus, any of the above-describedmonomer/building blocks may be used in this method.

Typically, the coupling is carried out in an aqueous buffer (pH 4-6,preferably 5) at a temperature below room temperature, e.g., about 0 or4° C. to about 15° C. The coupling may also be carried out in thepresence of a free radical initiator, under. e.g., sufficientultraviolet (UV) light to generate free radicals. Suitable wavelengthsof UV light that may be used include 300 to 450 nm. In some embodimentsa wavelength of about 365 nm is used. Examples of suitable free radicalinitiators include lithium acyl phosphinates and water soluble2,2-dimethoxy-2-phenylacetophenones.

To prepare trimers and larger oligomers, additional coupling steps maybe performed or multiple couplings may be performed simultaneously,depending on whether a linear or branched oligomer is desired. Thus, inone embodiment, the method the second monomer comprises a carboxyterminal alkenyl group and is coupled to a third monomer under freeradical conditions to form a thioether group and the third monomer isselected from a Ub or Ubl polypeptide including one or more cysteineresidues. Likewise, if the third monomer comprises a carboxy terminalalkene, an additional coupling to a fourth monomer comprising a cysteineis possible. In this way, linear oligomers of 4, 5, 6, 7, 8 or moremonomers may be constructed. Mixed linear oligomers may be formed if thesubsequent monomers have cysteines at different positions.Alternatively, the second monomer includes two cysteine residues, andthe second monomer is coupled to a third monomer under free radicalconditions to form a thioether group at each cysteine, wherein the thirdmonomer comprises a carboxy terminal alkene group. Subsequently furtheroligomers may be coupled to this branched oligomer as above.

In another aspect, a conjugate of the present oligomers with otherproteins is provided. Thus, the conjugates include an oligomer of Ub orUbl as described herein, covalently attached to a non-ubiquitin ornon-ubiquitin polypeptide. In such conjugates, the oligomer may beattached to the non-ubiquitin or non-ubiquitin-like polypeptide throughthe carboxy terminus of one of the monomers in the oligomer. Theoligomer may be attached to the non-Ub and non-Ubl usingenzyme-catalyzed ligation (see, e.g., C. M. Pickart and S. Raasi.“Controlled synthesis of polyubiquitin chains.” Meth. Enzymol. 2005,399, 21-36). Alternatively the same thiol-ene chemistry described forthe preparation of the oligomers themselves may be used to prepare theconjugate. Thus, site-directed mutagenesis of a lysine to a cysteine inthe protein of interest may be performed. Installation of the C-terminalalkene in an of the oligomers described herein is carried out asdescribed herein. Finally, the thiol-ene reaction between the oligomerand the mutated protein may be performed as described herein.

In another aspect, methods of using the oligomers are provided. Themethods include adding any of the oligomers described herein to culturedcells and determining the effects of the added oligomer(s) on thecultured cells. In some embodiments, the effects of the addedoligomer(s) are determined with respect to one or more of proteinexpression, mRNA levels, or levels of cellular signaling molecules.

EXAMPLES

The present technology is further illustrated by the following examples,which should not be construed as limiting in any way.

Example 1 Ubiquitin (Ub) Cloning and Expression

Cloning. Ubiquitin (1-76) (herein referred to as Ub₁₋₇₆) was purchasedfrom Addgene and cloned into pET-22b using the forward primerggcggtCATATGCAGATCTTCGTCAAG and reverse primerggcggtGCGGCCGTCAACCACCTCTTAGTCT containing NdeI and NotI restrictionssites. Lysine-to-cysteine mutations (KxC; where x is the position withinUb primary sequence) were introduced using the mutagenesis technique ofsplice overlap extension (SOE).¹ Primers containing the TGC mutationwere used to replace the respective codon for lysine. Aspartate 77 wasencoded in the reverse primer to afford the clone for UbD77.

Expression.

All Ub variants were expressed and purified from Rosetta™ 2(DE3)pLysScells (Novagen) following a procedure adapted from Pickart 2005.² Astarter culture was inoculated (10 mL LB media, 100 ug/mL Anmpicillin),grown to OD₆₀₀=0.5, and kept at 4° C. overnight. The starter culture wasadded to 1 L 2×YT media (16 g Peptone, 5 g NaCl, 10 g Yeast extract, 100mg/ml Ampicilin) and grown at 37° C. Cultures were then induced with 0.4mM IPTG at OD₆₀₀=0.6 and incubated for an additional 4 hours at 37° C.Cells were pelleted and resuspended in 150 mL lysis buffer (50 mM TrispH 7.5, 0.5 mM EDTA, 1 mM EgTA, 0.02% Igepal, 1 mM PMSF, 1 mM DTT).After sonication, the lysate was clarified by centrifugation at 8,000rpm for 30 min. Perchloric acid (70%, 0.19 mL) was added dropwise to thesoluble layer and stirred for 20 min to precipitate impurities. Aftercentrifuging at 8,000 rpm for 30 min, the supernatant was exchanged intoFPLC Buffer A (50 mM NH₄OAc pH 4.4, 1 mM EDTA, 1 mM DTT) with 2 roundsof dialysis (3.5 kD molecular weight cutoff snake-skin tubing). Ubvariants were further purified by cation exchange chromatography with agradient of 0% to 60% Buffer B (50 mM NH₄Oac pH 4.4, 1 mM EDTA, 1 mMDTT, 1 M NaCl) over 35 column volumes. Fractions containing Ub(monitored by SDS-PAGE) were combined, concentrated, exchanged into H₂Oand lyophilized: the purpose of which is to determine yields andminimize variation in the concentration of stock solutions.

Example 2 Yeast Ub Hydrolase 1 (YUH1) Expression and Purification

YUH1 in pET-3a was purchased from Addgene and expressed from Rosetta™2(DE3)pLysS cells (Novagen). A starter culture was inoculated (10 mL LBmedia, 100 ug/mL ampicillin), grown at 37° C. for 6 h, and stored at 4°C. overnight. 1 L of LB was inoculated with starter culture and grown toOD₆₀₀=0.8. A culture was induced with 1 mM IPTG and grown 13 h at 18° C.Cells were harvested by centrifugation (8,000×g at 4° C., 30 min) andpellet was resuspended in lysis buffer (20 mM NaPO₄, 0.5 M NaCl, pH7.4). Cells were lysed by sonication, and clarified by centrifugation(30,000×g at 4° C., 30 min). YUH1 was purified by ammonium sulfateprecipitation. The 40% and 60% ammonium sulfate fraction was dialyzedinto 25 mM NaCl, 50 mM HEPES pH 6.8, 1 mM DTT, and further purified byanion exchange (Buffer A: 25 mM Tris pH 8; Buffer B: 25 mM Tris pH 8, 1M NaCl; 0-100% B, 30 column volumes).

Example 3 Synthesis of Ub Allyl Amine Adduct (Ub-AA)

UbD77 (185.6 mg, 21.7 μM) was dissolved in a buffer containing 50 mMHepes pH 8, 1 mM EDTA, 30% DMSO, and 250 mM allylamine to a totalreaction volume of 25 mL. To this mixture was added YUH1 (25 nM). Aftertwo hours of shaking at room temperature, the reaction mixture wasquenched with TFA to a pH of 1-2, exchanged into Buffer A (50 mM NH₄OacpH 4.4, 1 mM DTT, 0.5 mM EDTA) and purified by cation exchangechromatography using the same method as in the Ub purification describedabove. Fractions containing Ub-AA were verified by MALDI massspectrometry, combined, concentrated, exchanged into water, andlyophilized for use in thiol-ene reactions. Final characterization wasdone by high resolution Fourier transform ion cyclotron resonance(FT-ICR) mass spectrometry using the methods explained below (FIG. 1).

Example 4 Synthesis of the Lithium Acyl Phosphinate (LAP) Free-RadicalPhotoinitiator

Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was synthesizedaccording to the procedures described by Majima³ and Fairbanks⁴ withoutmodifications (Scheme 1).

Example 5 Thiol-Ene Coupling (TEC) Reactions

Reaction Procedure.

Typical TEC reactions contained UbKxC (1 mM), Ub-AA (1 mM), LAP (0.5 mM)in 250 mM NaOAc buffer pH 5.1 (50 μL reaction volume). Samples wereplaced on ice and irradiated from above with 365 nM light for 30 minusing an OmniCure series 1500 light source placed 15 cm away from thesample. Control reactions contained wild type Ub (1 mM) instead ofUb-AA. TEC reactions were performed on all seven single UbKxC mutants(x=6, 11, 27, 29, 33, 48, 63) and analyzed by Coomassie-stained SDS-PAGE(FIG. 2). Control reactions omitting each reaction component show dimerdependence on all reaction components (FIG. 3). The same procedure wasperformed with the K6C, K48C; K11C, K48C; and K48C, K63C Ub doublemutants, but in this case Ub-AA (2 mM) was used to furnish therespective branched trimers.

Purification Procedure for Ub Dimers and Trimers.

Multiple TEC reactions for each dimer (K6C-, K48C-, and K63C-linked) andbranched trimer (K6C, K48C-; K11C, K48C-; and K48C. K63C-linked) werecombined (15×50 μL) and purified by cation exchange chromatography witha gradient of 0-60% B over 35 column volumes (FIG. 4).

Example 6 MS Analysis of Intact Full-Length Ub Dimers and Trimers

General Procedure.

Crude TEC reactions for all KxC-linked Ub dimers were reduced withdithiothreitol (DTT) then desalted using Amicon 3.5 kD MW cutofffilters. Samples were dissolved in a solution of water/MeCN/acetic acid(45:45:10) and injected into a 7T linear ion trap/Fourier transform ioncyclotron resonance (LTQ/FT-ICR) hybrid mass spectrometer (ThermoScientific Inc., Bremen, Germany) equipped with an automated chip-basednanoESI source (Triversa NanoMate, Advion BioSciences, Ithaca, N.Y.) asdescribed previously.⁵ The resolving power of the FT-ICR mass analyzerwas set at 200,000. All FT-ICR spectra were processed with in-housedeveloped Software (MadTHRASH version 1.0) using a signal to noisethreshold of 3 and fit factor of 60%, and then validated manually.

FIG. 5 shows the high resolution FT-ICR MS analysis of crude TECreactions using intact full-length proteins. The wide view showsabundance of Ub dimers in comparison to the starting materials UbKxC andUb-AA (M¹⁰⁺ charge state for starting materials, M²⁺ charge state fordimer is shown), while the zoom in shows each dimer compared to thetheoretical isotopic distribution (dots above peaks). FIG. 6 shows thehigh resolution FT-ICR MS analysis of each purified dimer. Circlesrepresent theoretical isotopic abundance distribution of the isotopomerpeaks. Calc'd: calculated most abundant molecular weight. Expt'l:experimental most abundant molecular weight. Similarly, FIG. 7 shows theFT-ICR analysis of each purified branched trimer.

Example 7 Electron Capture Dissociation (ECD) Analysis of theN□-Gly-L-homothiaLys Linkage

General Procedure for ECD.

For tandem mass spectrometry (MS/MS) experiments using ECD, individualcharge states of protein molecular ions were first isolated. Then, theions were dissociated by ECD using 3% “electron energy” and a 70 msduration with no delay. All FT-ICR spectra were processed with in-housedeveloped Software (MadTHRASH version 1.0) using a signal to noisethreshold of 3 and fit factor of 60%, and then validated manually. Theresulting mass lists were further assigned based on the protein sequenceof Ub with or without the modification (GlyGly-AA) at each Cys using a10 and 20 ppm tolerance for precursor and fragment ions, respectively.All reported M_(r) values are most abundant molecular weights.

Sample Preparation for ECD Analysis.

Purified K6C-, K48C-, and K63C-linked Ub dimers and branched Ub trimers(K6C, K48C-; K11C, K48C-; and K48C/K63C-linked) were minimally digestedwith trypsin (3 h digestion, 1:100 trypsin:Ub ratio) for detailedanalysis of the Nε-Gly-L-homothiaLys thioether linkage. Since trypsincleaves at position-74 of Ub, the products in this case are single Ubunits missing the C-terminal GlyGly motif, but attached to GlyGly-AA atthe respective cysteine residue (the product of this minimaltrypsinolysis is referred to as Ub₁₋₇₄GlyGly-AA): this greatlysimplifies analysis by ECD fragmentation (Scheme 2).

Detailed procedure for ECD analysis. Ub₁₋₇₄GlyGly-AA was fragmented byECD, and the resulting fragments were analyzed to verify the linkage atthe desired cysteine. Analysis was performed using in-house dataanalysis software (described above). The Ub₁₋₇₄ sequence was used topredict fragment molecular weights of all possible c ions (N-terminalions, numbered from amino acid 1) and z* ions (C-terminal ions numberedstarting from 1 from the C-terminal arginine and counting in reverse ofthe conventional amino acid numbering scheme). Raw data were analyzed tofind molecular weights of each observed fragments. Observed fragmentsand predicted fragments are compared to assign ion type to the observedpeaks. Ion assignments were then verified and analyzed with and withoutinclusion of a cysteine modification in the theoretical peakpredictions. When the thiol-ene modification was not taken into accountin the analysis, c ions after the cysteine and z* ions before thecysteine were lacking. Upon addition of 171 amu, which represents theGlyGly-AA motif, to the molecular weight of cysteine the ion types inobserved data were reassigned. In this case, the c and z* ions were bothpresent throughout the entire sequence. This data supports themodification at the desired cysteine residue and holds true for allmutants analyzed.

FIG. 8 shows the ECD analysis of K63C-linked dimer. FIG. 8A showsK63C-linked Ub₁₋₇₄ GlyGly-AA parent ion isolation (M⁹⁺ charge state)with insert of isotopomers. FIG. 8B is a map of observed fragments. Dataanalysis for the map on top includes Nε-Gly-L-homothiaLys thioetherlinker modification at cysteine-63 (red) in c and z* ion predictions.Bottom map does not include thioether linker modification in theoreticalanalysis. FIG. 8C shows the key ECD fragment ions for mapping thioetherlinkage site on UbK63C. Circles represent theoretical isotopic abundancedistribution of the isotopomer peaks. Calc'd: calculated most abundantmolecular weight. Expt'l: experimental most abundant molecular weight.

Similarly, FIG. 9 shows the ECD analysis of K6C-linked dimer, with FIG.9A being the K6C-linked Ub₁₋₇₄ GlyGly-AA parent ion isolation (M¹⁰⁺charge state) with insert of isotopomers. FIG. 9B is a map of observedfragments. Data analysis for the map on top includesNε-Gly-L-homothiaLys thioether linker modification at cysteine-6 (red)in c and z* ion predictions. The bottom map does not include thioetherlinker modification in theoretical analysis. FIG. 9C shows the key ECDfragment ions for mapping thioether linkage site on UbK6C, where circlesrepresent theoretical isotopic abundance distribution of the isotopomerpeaks.

FIG. 10 is the ECD analysis of K48C-linked Ub dimer. FIG. 10A isK48C-linked Ub₁₋₇₄ GlyGly-AA parent ion isolation (M⁹⁺ charge state)with insert of isotopomers, and FIG. 10B is the map of observedfragments. Data analysis for the map on top in FIG. 10B includesNε-Gly-L-homothiaLys thioether linker modification at cysteine-48 (red)in c and z* ion predictions, while the bottom map does not includethioether linker modification in theoretical analysis.

FIG. 11 is the ECD analysis of K6C, K48C-linked branched Ub trimer, withFIG. 11A K6C, K48C Ub₁₋₇₄ GlyGlyAA₂ parent ion isolation (M¹⁰⁺ chargestate) with insert of isotopomers and FIG. 11B a map of observedfragments. Data analysis includes Nε-Gly-L-homothiaLys thioether linkermodification at cysteine-6 and cysteine-48 (red) in c and z* ionpredictions. FIG. 12 shows the Key ECD fragment ions for K6C,K48C-linked trimer. Circles represent theoretical isotopic abundancedistribution of the isotopomer peaks. Calc'd: calculated most abundantmolecular weight. Expt'l: experimental most abundant molecular weight.

FIG. 13 shows the ECD analysis of K11C, K48C-linked trimer. FIG. 13A isK11C, K48C-linked Ub₁₋₇₄ GlyGlyAA₂ parent ion isolation (M¹⁰⁺ chargestate) with insert of isotopomers and FIG. 13B is a map of observedfragments. Data analysis for the map on the top in FIG. 13B includes theNε-Gly-L-homothiaLys thioether linker modification at cysteine-11 andcysteine-48 (red) in c and z* ion predictions. Bottom map does notinclude thioether linker modifications in the sequence. FIG. 14 showsthe key ECD fragment ions for K11C, K48C-linked trimer. Circlesrepresent theoretical isotopic abundance distribution of the isotopomerpeaks.

FIG. 15 is the ECD analysis of K48C, K63C-linked trimer, with K48C,K63C-linked Ub₁₋₇₄ GlyGlyAA₂ parent ion isolation (M¹⁰⁺ charge state)with insert of isotopomers (FIG. 15A) and the map of observed fragments(FIG. 15B). Data analysis in FIG. 15B for the map on top includes theNε-Gly-L-homothiaLys thioether linker modification at cysteine-48 andcysteine-63 (red) in c and z* ion predictions, while the bottom map doesnot include thioether linker modifications in theoretical analysis. FIG.16 shows the key ECD fragment ions for K48C, K63C-linked trimer, wherecircles represent theoretical isotopic abundance distribution of theisotopomer peaks.

Example 8 Optimization of Difficult Linkages (K27C, K29C, K33C)

TEC reactions were performed with varying amounts of Ub-AA and analyzedby high resolution FT-ICR MS. Relative amounts of product can becompared between each spectrum because the amount of UbKxC was the samein each reaction and can therefore be used as an internal standard(FIGS. 17-19).

Addition of Phosphinate Portion of LAP to Ub-AA.

Mass spectra of crude reaction mixtures shows a peak that corresponds tothe mass of Ub-AA plus the phosphinate portion of the LAP photoinitiator(see FIGS. 17-19). This observation has precedent from the work ofJockusch and Turro,⁶ which describes the rapid addition (k˜10⁷ M⁻¹s⁻¹)of phosphinoyl radicals to acrylates. Based on this work we propose theprocess shown in Scheme 3 occurs.

Example 9 DUB-Catalyzed Hydrolysis of Ub Dimers and Trimers

General Procedure.

IsoT and A20 were purchased from Boston Biochem, while AMSH waspurchased from LifeSensors. For the DUB-catalyzed hydrolysis of Ubdimers, reactions contained a particular KxC-linked Ub dimer (5 μM) andthe DUB (5 μM AMSH or 500 nM A20-OTU) in the DUB reaction buffer (50 mMTris pH 7.6, 25 mM KCl, 5 mM MgCl₂, 1 mM DTT) at 37° C. The DUB wasadded to the reaction last to initiate hydrolysis. At the time pointsindicated, 10 μL aliquots were taken and mixed with 3 μL of 6× Laemmlisample loading buffer. Samples were subjected to SDS-PAGE analysis andvisualized using silver stain. For the DUB-catalyzed hydrolysis of Ubtrimers, reactions contained Ub trimer (20 μM) and the DUB (5 μM AMSH,500 nM A20-OTU, or 1 mM IsoT) in the DUB reaction buffer at 37° C. Atthe time points indicated, 10 μL aliquots were taken and mixed with 3 Lof 6× Laemmli sample loading buffer. Samples were subjected to SDS-PAGEanalysis and detected by western blot using anti-ubiquitin antibody(P4D1) from Cell Signaling Technologies.

In particular, K11C. K48C. K48C, K63C, and K6C, K48C-linked trimers werechosen to systematically investigate the influence of an additional Ubunit on the hydrolysis of the K48C-linkage. IsoT efficiently processedall three trimers as evidenced by Western blot analysis (FIGS. 20-22).The most striking result, however, came while studying A20-OTU-catalyzedcleavage. That is, Western blot analysis indicated A20-OTU cleaved theK48C-linkage in K11C, K48C and K48C, K63C-linked trimers, whereas thesame linkage remained intact in the K6C, K48C-linked trimer. Since othernonselective DUBs such as those in the USP (Ubspecific protease) family,in particular USP7, trim K6C, K48C-linked Ub3 down to the monomer (seebelow and FIG. 23), the results with A20-OTU suggest the additional Ubunit appended to position-6 abrogates hydrolytic cleavage by K48-linkageselective DUBs. In the context of other linkage selective DUBs such asAMSH, the presence of a Ub appendage at position 48 does not influencecleavage of the K63C-linkage as indicated by the formation of Ub2 and Ubupon hydrolysis of the K48C, K63C-linked trimer (FIG. 21).

Our systematic examination of branched trimer topologies suggests thatbranch points in a polyUb chain furnish a regulatory mechanism forlinkage-selective interactions. Consistent with this analysis,K6-linkages are proposed to suppress degradation of target proteins by26S proteasomes. In principle, this could lead to the accumulation, andpossibly aggregation, of the target protein, which, in turn, would setthe stage for clearance by the lysosomal pathway. If the latter iseither unable or slow to process the aggregated proteins bearing polyUbchains, then toxic levels may begin to accrue in the cell: this is ahallmark of many neurodegenerative diseases. Interestingly, mixed K6-,K11-, and K48-linked polyUb chains have been observed in Tau aggregatesisolated from brain tissue of individuals with Alzheimer's disease.

To provide additional support for the hydrolytic cleavage of K6C.K48C-linked branched tri-Ub with DUBs lacking linkage selectivity, weinvestigated the activity of USP7. USP7 is a member of theubiquitin-specific protease (USP) family, and recently Sixnma andco-workers reported that the majority of isopeptidases in this familydisplay little linkage selectivity.⁷ The reason for specificallyinvestigating the activity of USP7 towards the K6C, K48C-linked trimeris that Ciechanover and co-workers demonstrated the regulation of RING1Bby USP7.⁸ Autoubiquitylation of RING B generates a putative branchedpolyUb chain linked through K6, K27, and K48.⁹ We surmised that if abranched chain containing K6- and K48-linkages is indeed attached toRING1B and USP7 is responsible for removing this chain, then USP7 shouldprocess K6C, K48C-linked tri-Ub. As shown in FIG. 23, dimeric andmonomeric Ub products are immediately produced upon treatment of thebranched chain with USP7.

Example 10 Preparation of Carboxy Terminus Modified Ubiquitin

C-Terminal Modification of Ubiquitin.

Reactions were performed at room temperature in buffer (50 mM HEPES pH8.0, 1 mM EDTA) containing 30% DMSO, 0.7 mM Ubiquitin D77, 250 mM ofindicated amine, and 250 nM YUH1. The reactions were initiated byaddition of YUH1, allowed to proceed for 12 hours, and quenched with 1%TFA. Modifications were analyzed using MALDI.

Calc'd Actual mass Ub mass Ub Amine Structure MW g/m conjugate conjugateallylamine

 57.1 8717.1 butylamine

 73.1 8733.1 8734.0 benzylamine

107.2 8767.2 8768.4 (boc)ethylene diamine

160.2 8820.2 8820.0 2-[2-((6-Chlorohexyl) oxy)ethoxy)ethanamine

223.7 8883.7 8884.6

REFERENCES

-   (1) Ho, S. N.; Hunt, H. D.; Horton, R. M.; Pullen, J. K.;    Pease, L. R. Gene 1989, 77, 51.-   (2) Pickart, C. M.; Raasi, S. Meth. Enzymol. 2005, 399, 21.-   (3) Majima, T.; Schnabel, W.; Weber, W. Makromol. Chem. 1991, 192,    2307.-   (4) Fairbanks, B. D.; Schwartz, M. P.; Bowman, C. N.; Anseth, K. S.    Biomaterials 2009, 30, 6702.-   (5) (a) Ayaz-Guner, S.; Zhang, J.; Li, L.; Walker, J. W.; Ge, Y.    Biochemistry 2009, 48, 8161. (b) Ge, Y.; Rybakova, I. N.; Xu, Q.;    Moss, R. L. Proc. Natl. Acad. Sci. USA 2009, 106, 12658. (c) Zhang,    J.; Guy, M. J.; Norman, H. S.; Chen, Y. C.; Xu, Q. G.; Dong, X. T.;    Guner, H.; Wang, S. J.; Kohmoto, T.; Young, K. H.; Moss. R. L.;    Ge, Y. J. Proteome Res. 2011, 10, 4054.-   (6) Jockusch, S.; Turro, N. J. J. Am. Chem. Soc. 1998, 120, 11773.-   (7) Faesen, A. C.; Luna-Vargas, M. P.; Geurink, P. P.; Clerici, M.;    Merkx, R.; van Dijk, W. J.; Hameed, D. S.; El Oualid, F.; Ovaa, H.;    Sixma, T. K. Chem. Biol. 2011, 18, 1550.-   (8) de Bie, P.; Zaaroor-Regev, D.; Ciechanover, A. Biochem. Biophys.    Res. Commun. 2010, 400, 389.-   (9) Ben-Saadon. R.; Zaaroor, D.; Ziv, T.; Ciechanover, A. Mol. Cell    2006, 24, 701.

EQUIVALENTS

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and apparatuses within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. Thus, for example, a group having 1-3 cells refers to groupshaving 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers togroups having 1, 2, 3, 4, or 5 cells, and so forth.

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. An oligomer comprising two or more monomers wherein each monomer is independently selected from a ubiquitin polypeptide or a ubiquitin-like polypeptide, and the monomers are covalently linked to each other via a thioether group or groups.
 2. The oligomer of claim 1 wherein each thioether group links the peptide backbone of one monomer to the carboxy terminus of another monomer.
 3. The oligomer of claim 1 wherein each thioether group comprises a cysteine residue of one of the monomers.
 4. The oligomer of claim 3 wherein each monomer is a mutant in which each cysteine residue in the thioether group replaces a lysine residue in the native sequence of the monomer.
 5. The oligomer of claim 1 comprising 2, 3, 4, 5, 6, 7, 8, 9 or 10 monomers.
 6. The oligomer of claim 1 wherein the oligomer is linear.
 7. The oligomer of claim 1 wherein the oligomer is branched.
 8. The oligomer of claim 7 wherein at least one monomer is covalently linked to at least 2 other monomers via thioether groups to the peptide backbone of the at least one monomer.
 9. The oligomer of claim 1 wherein the thioether groups of at least two monomers comprise cysteines at different positions within the monomer.
 10. The oligomer of claim 9 wherein at least one monomer comprises a thioether group at K6C and a thioether group at K48C, or at K11C and K48C, or at K48C and K63C.
 11. The oligomer of claim 1 wherein each monomer is independently selected from a polypeptide that a. has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, and 5; b. comprises a sequence that has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, and 5; or c. is a fragment of a polypeptide having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, and 5; wherein the ubiquitin or ubiquitin-like polypeptide is a substrate for a deubiquinating enzyme or for proteins containing ubiquitin-binding domains when covalently attached via its carboxy terminus to another protein.
 12. A conjugate comprising an oligomer of claim 1 covalently attached to a non-ubiquitin or non-ubiquitin-like polypeptide.
 13. The conjugate of claim 12 wherein the oligomer is attached to the non-ubiquitin or non-ubiquitin-like polypeptide through the carboxy terminus of one of the monomers in the oligomer.
 14. A ubiquitin or ubiquitin-like polypeptide comprising a carboxy terminal alkenyl group, wherein a cysteine residue replaces at least one lysine residue in the ubiquitin or ubiquitin-like polypeptide.
 15. The ubiquitin or ubiquitin-like polypeptide of claim 14 wherein the polypeptide a. has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, and 5; b. comprises a sequence that has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, and 5; or c. is a fragment of a polypeptide having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, and 5; wherein the ubiquitin or ubiquitin-like polypeptide is a substrate for a deubiquinating enzyme when covalently attached via its carboxy terminus to another protein.
 16. The ubiquitin or ubiquitin-like polypeptide of claim 14 wherein each of 1, 2 or 3 lysine residues of the native sequence is replaced with a cysteine residue.
 17. The ubiquitin or ubiquitin-like polypeptide of claim 14 comprising 1, 2, or 3 mutations selected from K6C, K11C, K27C, K29C, K33C, K48C, and K63C.
 18. The ubiquitin or ubiquitin-like polypeptide of claim 14 wherein the carboxy terminal alkenyl group has the structure: —NR¹(R²)CH═CH₂, wherein R¹ is H or C1-4 alkyl group; and R² is a substituted or unsubstituted alkylene, alkylene oxide, arylene or aralkylene group.
 19. The ubiquitin or ubiquitin-like polypeptide of claim 18 wherein the carboxy terminal alkenyl group has the structure: —NH(CH₂)_(n)CH═CH₂, wherein n is an integer from 0 to
 10. 20. A method of making an oligomer of claim 1 comprising coupling a first monomer to a second monomer under free radical conditions such that the first and second monomers are linked by a thioether group, wherein the first monomer is selected from a ubiquitin or ubiquitin-like polypeptide comprising a carboxy terminal alkenyl group and the second monomer is selected from a ubiquitin or ubiquitin-like polypeptide comprising one or more cysteine residues.
 21. The method of claim 20 wherein the ubiquitin or ubiquitin polypeptide a. has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, and 5; b. comprises a sequence that has at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, and 5; or c. is a fragment of a polypeptide having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS:1, 2, 3, 4, and 5, wherein the ubiquitin or ubiquitin-like polypeptide is a substrate for a deubiquinating enzyme when covalently attached via its carboxy terminus to another protein.
 22. The method of claim 20 wherein the coupling is carried out in the presence of a free radical initiator.
 23. The method of claim 22 wherein the coupling is carried out under sufficient ultraviolet light to generate fee radicals.
 24. The method of claim 22 wherein the fee radical initiator is selected from a lithium acyl phosphinate or a water soluble 2,2-dimethoxy-2-phenylacetophenone.
 25. The method of claim 20 wherein the second monomer comprises a carboxy terminal alkenyl group and is coupled to a third monomer under free radical conditions to form a thioether group and the third monomer is selected from a ubiquitin or ubiquitin-like polypeptide comprising one or more cysteine residues.
 26. The method of claim 20 wherein the second monomer comprises two cysteine residues and the second monomer is coupled to a third monomer under free radical conditions to form a thioether group, wherein the third monomer comprises a carboxy terminal alkene group. 